Phosphatidylinositol-3,4,5-trisphosphate (PIP3), one of the lipid second messengers, is present in cell membranes and plays an important role in intracellular signal transduction. To be more specific, it has been known to activate its binding protein such as Akt, PDK1 and Btk, and to adjust various cell functions associated with apoptosis, diabetes mellitus, cancer, and so on (Cantley, L. C. (2002) Science, 296, 1655-1657; Czech, M. P. (2000) Cell, 100, 603-606; Vanhaesebroeck, B and Alessi, D. R. (2000) Biochem. J., 346, 561-576). It has been clarified that production of PIP3 in cell membranes is catalyzed by phosphatidylinositol-3-kinase (PI3K) (Wymann, M. P. and Pirola, L. (1998) Biochim. Biophys. Acta, 1436, 127-150). A large number of stimuli elicit the PI3K activation, however, exactly how, when, and where the PIP3 production occurs has remained unknown. This appears to be due in part to the lack of appropriate methods to quantitatively analyze the spatial and temporal dynamics of PIP3 in single living cells. Actually, labeling of cells with [32P]orthophosphate has widely been used to measure PIP3 changes, however, this method has several limitations to obtain such spatial and temporal information, because millions of cells must be smashed and analyzed to obtain sufficient radiochemical signals. Recently, fused proteins of green fluorescent protein (GFP) and PIP3 binding domains derived from Btk (Varnal, P., Rother, K. I. and Balla, T. (1999) J. Biol. Chem., 274, 10983-10989), GRP1 (Venkateswarlu, K., Gunn-Moore, F., Tavare, J. M. and Cullen, P. J. (1998) Biochem. J., 335, 139-146), ARNO (Venkateswarlu, K., Oatey, P. B., Tavare, J. M. and Cullen, P. J. (1998) Curr. Biol., 8, 463-466) or Akt (Watton, J. and Downward, J. (1999) Curr. Biol., 433-436) have been reported as indicators for PIP3 accumulation in the cellular membrane, in which the translocation of the fusion proteins from the cytosol to the membrane has been explained to reflect the PIP3 accumulation. However, several factors such as changes in the cell shapes and membrane ruffles, which are frequently observed during fluorescence imaging experiments, cause serious artifacts. Moreover, it is difficult with these fluorescent fusion proteins to distinguish to which membranes the fusion proteins translocated in the cell.
The invention of the present application has been conducted in view of the above-mentioned circumstances and its object is to solve the problems in prior arts. The present invention aims to provide a probe for quantitatively detecting when and where lipid second messengers such as PIP3 are produced in single living cells. The invention of the present application also provides a method for screening a substance which affects the signaling by an intracellular lipid second messenger and a diagnostic method by measuring the signal associated with the diseases by using the probe as such.